CN115825201A - Method for ionizing and reducing protein disulfide bonds and determining free thiol ratio by using capillary electrophoresis apparatus - Google Patents
Method for ionizing and reducing protein disulfide bonds and determining free thiol ratio by using capillary electrophoresis apparatus Download PDFInfo
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Abstract
The invention belongs to the technical field of protein or polypeptide structure determination, and particularly relates to a method for ionizing and reducing a protein disulfide bond and determining the proportion of free sulfydryl by using a capillary electrophoresis apparatus. The method comprises the following steps: 1) A free mercapto alkylation step; 2) Enzyme digestion; 3) Reducing the enzyme digestion product and then alkylating; 4) And (5) analyzing and detecting. The analysis method provided by the invention can simultaneously and quantitatively detect the free sulfydryl ratio of each cysteine in the protein in one test, and provides a new method for quantitatively detecting the disulfide bonds of the protein.
Description
Technical Field
The invention belongs to the technical field of protein or polypeptide structure determination, and particularly relates to a method for ionizing and reducing a protein disulfide bond and determining the proportion of free sulfydryl by using a capillary electrophoresis apparatus.
Background
Disulfide bonds are covalent bonds in the form of "-S-" formed by covalent crosslinking of two cysteine side chain thiol (-SH) groups within or between proteins or polypeptide chains, are a very critical protein structure, and play a role in maintaining structural stability in many protein molecules. In the biomedical field, more and more proteins or polypeptides are used as biologics or vaccines for the treatment of a wide variety of diseases. These biological agents include hormones, growth factors, monoclonal antibodies, etc., and most of these biological molecules contain disulfide bonds. Disulfide bond-containing drugs are susceptible to disulfide bond exchange and rearrangement during synthesis, and these side reactions can lead to the formation of non-native disulfide bonds, aggregation of proteins and possibly further induce immunogenicity, and thus inactivate the drug. Therefore, the localization and analysis of disulfide bonds in polypeptide and protein drugs is one of the important research points in the biomedical field. The development of peptide and protein drugs is rapid, and the characterization of disulfide bonds has become an indispensable important link in the evaluation of the safety and effectiveness quality of biopharmaceuticals.
In the process of protein biological drug characterization, it is usually necessary to detect whether disulfide bonds of a sample protein are formed or are still in a free thiol state, and what proportion of the disulfide bonds become free thiols.
In order to quantitatively detect the free sulfydryl in the sample, the content of the free sulfydryl is generally analyzed and detected by 5,5' -dithio-bis- [ 2-nitrobenzoic acid ] in the prior art. Then, the sample is cut into peptide fragments by enzyme in a non-reduction state, and finally, the enzyme-cut products are analyzed by LC-MS (liquid chromatography-mass spectrometry) by using liquid substances to obtain the positions of disulfide bonds. However, this method can only obtain the free thiol ratio of all cysteines as a whole, and cannot obtain the free thiol ratio of each cysteine in the sample.
The invention patent CN 111381048A is a technology that two alkylating reagents are used successively to mark free sulfydryl and sulfydryl after disulfide bond reduction, then peptide segments containing the two are distinguished by mass spectrum, and mass spectrum peak area quantification is used, but during reduction, chemical reagent dimercaptothreitol is used for disulfide bond reduction. But dimercaptothreitol is used as a chemical reduction reagent to always interfere with subsequent liquid chromatography, and sometimes can react with disulfide bonds to form sulfydryl, so that reaction derivatives are produced, and data analysis is interfered. In addition, the reaction time is usually more than 30 minutes, which is not convenient enough.
For the above reasons, it is desirable to provide a method for rapidly and quantitatively detecting the ratio of free thiol groups in a protein or polypeptide sample.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for quickly and accurately analyzing the content of cysteine free sulfydryl in protein or polypeptide.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme:
a method for ionizing and reducing a disulfide bond of a protein and determining a free thiol ratio thereof using a capillary electrophoresis apparatus, comprising the steps of:
1) A free mercapto alkylation step: under the condition that a sample is not treated, adding an alkylating reagent 1 which can generate irreversible reaction with the cysteine free sulfydryl originally existing in the sample to obtain an alkylated product 1; this step has two functions, one is to label the free thiol group to distinguish the disulfide-bond forming thiol group; and secondly, the free sulfhydryl group is blocked, so that the free sulfhydryl group can not form mismatch with other sulfhydryl groups which attack the formed disulfide bond in the subsequent sample treatment process, thereby interfering the detection.
2) Enzyme digestion step: adding protease into the alkylation product 1, and carrying out enzymolysis on the alkylation product 1 to obtain peptide fragments, wherein each peptide fragment is required to contain at most one pair of disulfide bonds;
3) And (3) carrying out alkylation after enzyme digestion product ionization reduction: adding an alkylating reagent 2 into the enzyme digestion product, and ionizing and reducing all disulfide bonds in the sample into free sulfydryl by using a capillary electrophoresis apparatus to obtain an alkylation product 2; the alkylating agent 2 and alkylating agent 1 are different in molecular weight;
the operation of the step has the following functions: the added alkylating reagent 2 needs to be distinguishable from alkylating reagent 1 in molecular weight so that free thiols can be distinguished from disulfide thiols in subsequent mass spectrometry analysis.
4) And (3) analyzing and detecting: and taking the sample out of the ionization capillary by the pressure of the capillary electrophoresis apparatus, centrifuging, taking supernatant, performing analysis data acquisition on the supernatant by using a liquid chromatograph-mass spectrometer, and analyzing the data.
Preferably, for the step of assaying, said step of assaying comprises a step of determining the proportion of free thiols of cysteine, which comprises the following steps of assaying: obtaining mass spectrum peak areas of all cysteine-containing peptide fragments according to a liquid chromatograph-mass spectrometer, and for a specific cysteine, taking the sum of mass spectrum peak areas of cysteine peptide fragments marked by an alkylating reagent 1 in a reduction sample as a molecule, and taking the sum of mass spectrum peak areas of cysteine peptide fragments marked by the alkylating reagent 1 or the alkylating reagent 2 as a denominator, wherein the obtained percentage data is the free thiol ratio of the cysteine at the position.
Preferably, alkylating agents with different molecular weights are selected in step 1) and step 3), and the alkylating agent is one of the following ranges: 1-cyano-4-dimethylaminopyridine tetrafluoroborate, N-ethylmaleimide, biotin-maleimide, iodoacetamide or iodoacetic acid.
Preferably, the concentration of the polypeptide sample in step 1) is 1-20mg/mL.
Preferably, the reaction conditions of the alkylation reaction in step 1) and step 3) are: the reaction temperature is 4-45 ℃; the reaction time is 10min to 18h; the concentration of alkylating reagent 1 is 1-100mM.
In the present invention, for the selection of the protease in step 2), one of ordinary skill in the art can select the corresponding enzyme according to the characteristics of the protein to be analyzed, and by combining with the conventional knowledge in the art.
Preferably, the reduction method used in step 3) is capillary electrophoresis ionization without any chemical reduction reagent.
Further preferably, the reaction conditions of the reduction reaction in step 3) are: the diameter of the quartz bare bubbling capillary bare tube of the capillary tube is 100 mu m or more, and the length is 30-100cm; ionizing background buffer solution by 50% acetonitrile; 1% formic acid; the voltage is-15 v/cm of capillary length, and the time is 1-2 minutes; capillary length of 15v/cm, 1-2 minutes; a platinum electrode was used.
Compared with the prior art, the invention has the following beneficial effects:
(1) The analysis method provided by the invention can simultaneously and quantitatively detect the free sulfydryl ratio of each cysteine in the protein in one test, and provides a new method for quantitatively detecting the disulfide bonds of the protein.
(2) The analysis method provided by the invention is used for pairing the existing free sulfydryl before any treatment is carried out on the sample, so that the existing free sulfydryl is prevented from attacking other disulfide bonds during subsequent sample treatment and interfering the detection result;
(3) The idea of adopting the capillary electrophoresis apparatus to ionize the sample to reduce the disulfide bond is simple and quick to operate, and can avoid the interference of a chemical reagent on the subsequent mass spectrometry;
(4) The invention provides a method for calculating the mismatching proportion of disulfide bonds at a certain position and the free thiol proportion of each cysteine in protein by using a calculation formula based on mass spectrum peak area statistical data, and the method can be realized without complex detection equipment, is simple and quick to operate, and is beneficial to industrial production and application.
(5) Compared with the chemical reduction method, the method has no difference on the aspect of final results, but only saves time, improves efficiency and has no interference of other chemical substances.
Drawings
FIG. 1 is a LC-MS total ion flow diagram for a non-reducing sample;
FIG. 2 is a LC-MS total ion flow diagram of a reduced sample.
Detailed Description
The invention is further illustrated by the following examples. These examples are for illustrative purposes only and do not limit the scope and spirit of the present invention.
Example 1
A method for ionizing and reducing a disulfide bond of a protein and determining a ratio of free thiols thereof using a capillary electrophoresis apparatus, comprising the steps of:
1. adding 5mM N-ethylmaleimide NEM into 200 μ g interferon alpha 1b, dissolving with appropriate amount of water, and reacting at 4 deg.C overnight;
2. replacing the solution with a buffer containing 2M urea 100mM Tris-HCl, pH =7.5 using a 10kD semi-permeable membrane;
3. adding Glu-C protease according to the mass ratio of 1;
4. to the sample was added iodoacetamide at a final concentration of 40mM, acetonitrile μ L at a final concentration of 50% and formic acid at 1%.
5. The sample was subjected to reductive alkylation reagent 2 treatment: the sample was injected into a bare quartz bubbling capillary tube of 100 μm diameter and 50cm length using 50psi pressure; ionizing background buffer 50% acetonitrile; 1% formic acid; apply voltage-750 v capillary long, 2 minutes; +750v capillary long, 2 minutes; capillary electrophoresis apparatus requires platinum electrodes.
6. After the reaction is stopped, centrifuging the sample, taking the supernatant, and analyzing the sample supernatant by using a liquid chromatograph-mass spectrometer;
the chromatographic and mass spectrometric conditions used were as follows:
the liquid chromatography parameters were:
mobile phase a was 0.1% fa aqueous solution,
mobile phase B0.1% FA acetonitrile solution,
the chromatographic Column is Nano C18 Column,
1.7μm，2.1mm×150mm，
the temperature of the column oven is 60 ℃,
the temperature of the sample room is 4 ℃,
the flow rate was 0.3mL/min,
the detection wavelength is 214nm, and the detection wavelength,
the elution gradient was set as follows:
in the method, the enzymolysis product of the test sample is subjected to chromatographic separation by UPLC.
Parameters of mass spectrometry method
MS systems QE plus, thermo,
the ionization mode is ESI positive,
the scanning range of the primary mass spectrum is 300-2000Da,
the second-order mass spectrum scans in the range of 100-2000Da,
the capillary voltage is 2.5kV,
the voltage of the taper hole is 60V,
the ion source temperature was 120 c,
the temperature of the solvent gas is 400 ℃,
the air flow speed of the taper hole is 50L/h,
the desolvation gas flow rate is 800L/h.
The proportion of each cysteine free thiol group of interferon alpha 1b was analyzed using the above method.
Interferon alpha 1b has 166 amino acids, 4 cysteines, 2 disulfide bonds in its amino acid sequence. Theoretical disulfide bond position: C1-C99, C29-C139.
After the steps of sample processing, liquid quality data acquisition, data analysis and the like, the following analysis results are obtained:
the peptide fragments modified by iodoacetamide at C1, C29, C99 and C139 in the reduced sample are summarized in tables 1, 3, 5 and 7, and the peptide fragments modified by N-ethylmaleimide NEM are summarized in tables 2, 4, 6 and 8. Wherein, the cysteine modified by the iodoacetamide represents that the cysteine is previously involved in the formation of a disulfide bond, and the modification by the NEM represents that the cysteine is previously present in a free sulfhydryl form in a sample.
TABLE 1 data table of mass spectra of peptide fragments modified with iodoacetamide in C1
TABLE 2 Mass Spectrometry data sheet of peptide fragments modified with N-ethylmaleimide in C1
TABLE 3 Mass Spectrometry data Table of peptide fragments modified with iodoacetamide by C29
TABLE 4 Mass Spectrometry data Table of peptide fragments modified with N-ethylmaleimide in C29
TABLE 5 Mass Spectrometry data Table of peptide fragments with C99 modified by iodoacetamide
TABLE 6 Mass Spectrometry data Table of peptide fragments modified with N-ethylmaleimide in C99
TABLE 7 Mass Spectrometry data Table of peptide fragment with iodoacetamide modification by C139
TABLE 8 Mass Spectrometry data Table of peptide fragments modified with N-ethylmaleimide at C139
The calculation process and conclusion of the free sulfydryl ratio are as follows:
1. as can be seen from tables 1 and 2, the sum of the mass spectrum peak areas of the peptide fragment modified by iodoacetamide for C1 in the reduced sample is 799140137.68, and the sum of the mass spectrum peak areas of the peptide fragment modified by N-ethylmaleimide is 3762213.11. Therefore, the ratio of free mercapto groups of C1 is 3762213.11/(3762213.11 + 799140137.68) =0.47%.
2. As can be seen from tables 3 and 4, the sum of the mass spectrum peak areas of the peptide fragment modified with iodoacetamide for C29 in the reduced sample is 709352700.36, and the sum of the mass spectrum peak areas of the peptide fragment modified with N-ethylmaleimide is 1109540.31. Therefore, the ratio of free mercapto group of C29 is 1109540.31/(1109540.31 + 709352700.36) =0.16%.
3. As can be seen from tables 5 and 6, the sum of the mass spectrum peak areas of the peptide fragment modified with iodoacetamide at C99 in the reduced sample is 905314221.12, and the sum of the mass spectrum peak areas of the peptide fragment modified with N-ethylmaleimide is 2075599.45. Therefore, the ratio of free mercapto group of C99 is 2075599.45/(2075599.45 + 905314221.12) =0.23%.
4. As can be seen from tables 7 and 8, the sum of the mass spectrum peak areas of the peptide fragment modified with iodoacetamide for C139 in the reduced sample was 1171641811.26, and the sum of the mass spectrum peak areas of the peptide fragment modified with N-ethylmaleimide was 1583317.016. Therefore, the ratio of free mercapto group of C139 is 1583317.016/(1583317.016 + 1171641811.25781) =0.13%.
The ratio of free thiol groups of each cysteine is summarized as follows: c1 0.47%, C29.16%, C99.23%, C139.13%. I.e. most cysteines form disulfide bonds with other cysteines.
It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A method for ionizing and reducing a disulfide bond of a protein and determining a ratio of free thiols thereof using a capillary electrophoresis apparatus, comprising the steps of:
1) Free thiol alkylation step: adding an alkylating reagent 1 to react to obtain an alkylated product 1 under the condition that a sample is not treated;
2) Enzyme digestion step: adding protease into the alkylation product 1, and carrying out enzymolysis on the alkylation product 1 to obtain peptide fragments, wherein each peptide fragment is required to contain at most one pair of disulfide bonds;
3) And (3) alkylation step after enzyme digestion product reduction: adding the enzyme digestion product obtained in the step 2) into an alkylation product 2, ionizing by using a capillary electrophoresis apparatus to reduce the disulfide bond into free sulfydryl, and alkylating by an alkylating agent 2 to obtain an alkylation product 2;
4) And (3) analyzing and detecting: and (4) carrying out data acquisition on the two samples of the enzyme digestion product and the alkylation product 2 in the step 3) by using a liquid chromatograph-mass spectrometer, and carrying out data analysis on the acquired data.
2. The process of claim 1, wherein the alkylating agents of different molecular weights are selected from step 1) and step 3), and the alkylating agents are selected from one of the following ranges: 1-cyano-4-dimethylaminopyridine tetrafluoroborate, N-ethylmaleimide, biotin-maleimide, iodoacetamide, or iodoacetic acid.
3. The method of claim 1, wherein the alkylating agent used in step 1) is N-ethylmaleimide and the alkylating agent used in step 3) is iodoacetamide.
4. The method according to claim 1, wherein the concentration of the polypeptide sample in step 1) is 1-20mg/mL.
5. The method of claim 1, wherein the alkylation reaction in step 1) is carried out under the following reaction conditions: the reaction temperature is 4-45 ℃; the reaction time is 10min to 18h.
6. The method of claim 1, wherein the concentration of alkylating reagent 1 in step 1) is 1-100mM.
7. The method of claim 1, wherein the reaction conditions of the ionization reduction reaction in the step 3) are as follows: the diameter of the quartz bare bubbling capillary bare tube is 100 mu m or more; the length is 30-100cm; ionizing background buffer solution by 50% acetonitrile; 1% formic acid; the voltage is-15 v/cm of capillary length, and the time is 1-2 minutes; capillary length of 15v/cm, 1-2 minutes; a platinum electrode was used.
8. The method of claim 1, wherein the sample alkylation 2 reaction conditions are: the sample was reduced by adding 1-100mM alkylating reagent 2.
9. Use of the method according to claims 1-8 for the analysis of the free thiol content of a polypeptide or protein.
10. The method of claims 1-8, wherein the polypeptide is interferon alpha 1b and the protease is Glu-C protease.
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| CN110824096A (en) * | 2018-08-13 | 2020-02-21 | 齐鲁制药有限公司 | Method for analyzing protein mismatching disulfide bond |
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| CN111554345A (en) * | 2020-05-15 | 2020-08-18 | 吉林大学 | Method for constructing three-dimensional structure of protein based on specific cross-linked tyrosine |
| US20210010055A1 (en) * | 2018-03-16 | 2021-01-14 | Bristol-Myers Squibb Company | Metabolic enzyme activity and disulfide bond reduction during protein production |
| CN114786719A (en) * | 2019-10-02 | 2022-07-22 | 阿拉玛布治疗学股份有限公司 | Anti-connexin antibody formulations |
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2022
- 2022-07-28 CN CN202210903510.4A patent/CN115825201A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333281A (en) * | 2018-02-08 | 2018-07-27 | 吉林大学 | A method of measuring recombinant human granulocyte colony stimulating factor disulfide bond composition |
| US20210010055A1 (en) * | 2018-03-16 | 2021-01-14 | Bristol-Myers Squibb Company | Metabolic enzyme activity and disulfide bond reduction during protein production |
| CN110824096A (en) * | 2018-08-13 | 2020-02-21 | 齐鲁制药有限公司 | Method for analyzing protein mismatching disulfide bond |
| CN114786719A (en) * | 2019-10-02 | 2022-07-22 | 阿拉玛布治疗学股份有限公司 | Anti-connexin antibody formulations |
| CN111381048A (en) * | 2020-03-22 | 2020-07-07 | 烟台迈百瑞国际生物医药有限公司 | Analysis method for free sulfhydryl sites and ratio of antibody and application thereof |
| CN111554345A (en) * | 2020-05-15 | 2020-08-18 | 吉林大学 | Method for constructing three-dimensional structure of protein based on specific cross-linked tyrosine |
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